Prevalence of zoonotic intestinal helminths of canids in moghan plain, northwestern iran.

Background The present study was aimed to elucidate the status of intestinal helminth infections in canids of Moghan Plain, northwestern Iran. Methods Eighty-five intestine samples from dead or shot wild canids, 59 fecal samples from sheepdogs and 5 from red foxes were collected from 2006 to 2008 and examined in Parasitology department of Pasteur Institute of Iran. Results Generally, adult worms, larvae, and eggs of 13 species of various parasitic helminths were recovered. Necropsy examinations showed that 96.47% animals harbored at least one helminth species. The prevalence of different species in necropsy were Mesocestoides sp. 84.7%, Rictolaria spp. 55.3%, Macranthorhynchus hirudinaceus 45.9%, Toxocara canis 43.5%, Toxascaris spp. 35.3%, Joyeuxiella sp. 34.1%; hookworms; 22.4%, Taenia spp. 11.8%, Alaria spp. 2.4% and Dipylidium caninum 1.2%. Besides, eggs belonging to 10 species of parasitic helminths were identified in 46 fecal samples and generally, 30.9% of samples harbored eggs of at least one helminth species. Conclusion The high prevalence of various helminth infections among canids in Moghan plain and contamination of environment by helminths eggs may increase the risk of infection for native people.


Introduction
ild and domestic animals are considered as main sources of emerging human and domestic livestock pathogens and zoonoses of public health significance (1). In Iran, four categories of canids, namely feral or stray dogs, working sheepdogs, pet dogs and wild canids (especially red foxes and jackals) are known as reservoirs of numerous zoonotic diseases. There are some reports on prevalence of intestinal helminths among canids from different parts of Iran (2)(3)(4)(5)(6)(7)(8). Due to relatively high annual precipitation, building of dams on Aras River, huge irrigations networks and agriculture plans Moghan plain has become one of the most important areas for farming and stockbreeding in Iran, where nomadic, rural, and urban inhabitants come into close contacts with domestic and wild canids. There were two reports on the prevalence of helminthic infections in the carnivores of this area dating back to 1973 and 1993 (3,5). Since then, intensive economic and social alterations including rapid population growth and development of villages and cities have resulted to major ecological changes in Moghan Plain, Iran (9). The present study was conducted to elucidate the status of intestinal helminth infections among canids in this area.

Study area
This study was performed in the Moghan Plain (local name: Dasht-e-Moghan), situated in the northernmost parts of the Ardebil Province, northwestern Iran (Fig.  1). The area comprises three counties including Pars Abad, Bileh Savar and Germi covering an area of nearly 5245 km 2 with a total population of approximately 310,000. The area covers the low landing areas with altitude of 32m up to the plains of 1023 m high with mean annual precipitation of 223 mm. The area is bordered with Azerbaijan Republic to the north and east with longitudes and latitudes ranges of approximately 46°52'53″E -48°21'30″ E and 39° 0' 0″N -39°36'20″N, respectively. The inhabitants are mainly of Azeri ethnic group and mostly practice farming and stockbreeding.

Samples
From September 2006 to November 2008, 149 samples were collected from different localities in the study area. Eighty five intestine samples were obtained from dead or shot wild canids comprising 84 red foxes (Vuleps vulpes) and one golden jackal (Canis aureus). The animal carcasses were autopsied; small intestines were tied off at both ends and removed. The fecal samples were also collected from the animals' rectum as well. Besides, five environmentally deposited feces from vicinity of red foxes dens and 59 fecal samples from working sheepdogs (Canis lupus.familiaris) were collected and stored in 70% ethanol. All Samples were frozen at −80 °C for at least 5 days for safety reasons and then kept at −20 °C until used.

Intestinal Scraping Technique
Small intestines were opened with gut scissors and visually inspected for the presence of E. multilocularis and other helminths. After removal of coarse intestinal contents, smear samples were taken from locations at 10 cm intervals by scraping the mucosa with glass slides and then pressing it on Petri dishes. The samples were examined using a stereomicroscope with magnifications of 4X to 40X. All procedures were performed with appropriate safety measures taken at site. The numbers and developmental stages of the recovered helminths were recorded. Their morphological features were drawn and studied using a light microscope equipped with a Camera Lucida apparatus.

Sedimentation and Counting Technique
Small intestines were divided into four to six pieces of 20 cm length and opened longitudinally. Large helminths were removed directly, and then pieces of gut were immersed in normal saline and incubated at 37°C for 30 min. The mucosa was stripped between two pressed fingers. Twenty scrapes were also made with a spatula or microscope slides. Samples were then left to sediment for 20 min, and then washed two times or until the supernatant was clear. Amounts of 5-10 ml of precipitate were examined for parasites against a dark background in rectangular plastic dishes with a counting grid, using a stereomicroscope at 120X magnification. All collected parasites were stored in 70% alcohol (11).

Stool concentration methods
Formol-ether technique and sucrose floatation method were performed according to Muller (12) and Gary (13), respectively.
Statistical methods SPSS 16.0.0 (SPSS Incorporation) was used for statistical analysis. Pearson's Chi Square tests were performed to determine the probable relation between different variables.

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.7% of examined animals were infected with at least with one helminth infection, but according to necropsy examination of 85 animal intestines (84 red foxes and one jackal), 96.4% animals harbored at least one helminth infection (Table 1). Based on necropsy methods, the most common helminth infection among red foxes of Moghan plain belonged to Mesocestoides spp. with the prevalence as high as 79.8% (Table 2). 54.3% of animals were infected with two or more helminths. The most intensive case, a male fox in Bileh Savar, was infected with 7 helminth species (Fig 2). There was a significant difference among the prevalence of helminth infections in three counties (χ 2 = 21.973; df=2; P<0.001). All 36 samples (100%) from Bileh Savar County were infected with at least one helminth infection, whereas the rates in Pars Abad and Germi were 72.2% and 57.1%, respectively. A significant difference was also observed among the prevalence of helminth infections regarding the sampling seasons (χ 2 = 44.715; df=3; P<0.001).
All the samples collected during the spring-harbored eggs, larvae or adult forms of at least one helminth species, while in summer, autumn and winter the rates were 94.3%, 44.8% and 52.9%, respectively. The prevalence of Toxocara.canis, Mesocestoides sp., Rictolaria sp., Macranthorhynchus hirudinaceus, Hookworms and Joyeuxiella sp., infections differed significantly in different seasons.
All the helminths showed the highest and lowest prevalence in summer and winter (Table 3). There was a significant relation between all aforementioned infections and the location of sampling so that the prevalence of T. canis, Toxascaris sp., Mesocestoides sp., M. hirudinaceus and Joyeuxiella sp., infec-tions in Bileh Savar were higher than the two other counties ( Table 4).

Discussion
The result of this survey showed that the most common helminth infection belonged to Mesocestoides sp. This helminth has already been reported in jackals, red foxes, wolves, stray, and sheep dogs from different parts of Iran (2,4,5,8,14). The presence of this helminth has also been documented in other countries including Belarus, Portugal, and Italy with prevalence rates of 13.8%, 30.2%, and 26.6% respectively (15)(16)(17). However, in a study on 588 red foxes in Great Britain, no animal was shown to harbor this parasite (11). The prevalence of this helminth in our study was 79.6%, which was higher than all previous reports except one from western part of Iran (2). Intensity of the infection, determined by a subjective method, was high in a considerable number of animals so that 23.6% and 19.1% of samples were 3+ and 4+, respectively. The infection rates during the summer and in Bile Savar County were significantly higher than other seasons and counties. The higher prevalence of Mesocestoides sp. infection in this study, in comparison to the previous ones, may be due to food resources that are available for red foxes. It seems that coprophagic mites, as the first intermediate host, and amphibians, reptiles, small mammals and birds, especially home-breaded chickens, as the second intermediate hosts for parasite, are a major part of red foxes' diet.
In necropsy of red foxes, 44 out of 84 (52.4%) were infected with animal ascarids, the rate among domestic dogs was lower (32.2%) which might be due to type of samples (stool) and examination method. These worms are considered as the most important causative agents of visceral larva migrant (VLM) disease in humans (12). The prevalence of these worms in our study was higher than most of previous ones (2, 5, 6 14), even in domestic dogs in which stool samples were examined rather than intestines.  (11), Italy (15) and Portugal (16), which is probably resulted from different climatic conditions and animal fauna in these countries. In this study, Acanthocephala infection showed a high prevalence among red foxes as 46.6%. The acanthocephalan species was diagnosed as M. hirudinaceus based on morphologic features. In the previous reports, the prevalence of M. hirudinaceus was 10-23% (5, 2). In Belarus 3.2% and in Portugal 1.6% of red foxes were infected with Acanthocephalan species (16,17). However, in two separated studies in Great Britain and Italy, no acanthocephalan helminth was found (11,15 (11,17). As discussed earlier, the high prevalence of this worm may be a reflection of food sources, i.e. arthropods and entomophagous vertebrates that are available for red foxes in the area. Alaria sp. was the only parasitic trematode species that we could recover. Alaria sp. infections were previously reported from jackals and dog in Caspian Sea littoral (14), jackals in western provinces (2) and red foxes in this area (5). The prevalence of A. alata in Italy and Portugal showed to be 24.2% and 27.4%, respectively (15,16). The lower prevalence of Alaria spp. in Iran, compared to European countries may probably be due to less favored environmental condi-tions for growth and survival of snails that serve as first intermediate hosts.
The lower prevalence of Mesocestoides sp., Rictolaria spp., T. canis, Joyeuxiella sp., and Toxascaris spp. infections in Germi in comparison to Bileh Savar and Pars Abad is probably due to high altitude and cold climate condition. The other two counties are warmer and are commonly utilized as winter resorts by completely nomadic populations of Ardebil Province. The lower total prevalence of helminth infections in autumn and winter compared to spring and summer could be resulted from decreased populations of intermediate or paratenic hosts especially arthropods and lagomorphs in cold seasons on which the canids pray. This study showed that various helminthes, especially soil transmitted species and causative agents of cutaneous larva migrans (CLM) and VLM syndromes in humans are of high prevalence among canids of the Moghan plain. High humidity of the area resulting from relatively high annual precipitation (approximately 300 mm) and vicinity to Caspian Sea may provide a suitable environmental condition for growth and survival of eggs and larvae of the soil-transmitted helminths. Considering the high prevalence and intensity of the helminths infection among canids as well as numerous world reports on human cases, an investigation on medical and veterinary importance of these zoonotic infections in the area seems to be necessary.